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Probing off-target effect of LMNA siRNA and pro-siRNA in HeLa-d1EGFP cell line

ABSTRACT: We have develped a novel method of making siRNAs (named pro-siRNA for prokaryotic siRNA). To evaluate off-targeting of pro-siRNA, we compared mRNA expression profile of HeLa-d1EGFP cells transfected with 4 nM LMNA siRNAs and pro-siRNAs by microarray. We used microarray to study off-target effect of siRNAs in HeLa-d1EGFP cell line. After transfection of siRNAs for 24 hrs, RNA were extracted using Trizol. RNA samples were sent to Microarray Core at Dana-Farber Cancer Institute (http://macf-web.dfci.harvard.edu/index.php?option=com_frontpage&Itemid=1) to be probed using Human Gene 1.0 ST (Affymetrix).

ORGANISM(S): Homo sapiens

SUBMITTER: Linfeng Huang

PROVIDER: E-GEOD-44105 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Efficient and specific gene knockdown by small interfering RNAs produced in bacteria.

Huang Linfeng L Jin Jingmin J Deighan Padraig P Kiner Evgeny E McReynolds Larry L Lieberman Judy J

Nature biotechnology 20130310 4

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells and may be used for therapeutic purposes to knock down genes implicated in disease. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in Escherichia coli. This method relies on ectopic expression of p19, an siRNA-binding protein found in a plant RNA virus. When expressed in E. coli, p19 stabilizes an ...[more]

PMID: 23475073

Similar Datasets

Probing off-target effect of LMNA siRNA and pro-siRNA in HeLa-d1EGFP cell line

Project description:We have develped a novel method of making siRNAs (named pro-siRNA for prokaryotic siRNA). To evaluate off-targeting of pro-siRNA, we compared mRNA expression profile of HeLa-d1EGFP cells transfected with 4 nM LMNA siRNAs and pro-siRNAs by microarray.

2013-02-06 | GSE44105 | GEO

Probing the off-target effect of EGFP siRNA and pro-siRNA in the HeLa-d1EGFP cell line

2013-02-12 | GSE44256 | GEO

Probing the off-target effect of EGFP siRNA and pro-siRNA in the HeLa-d1EGFP cell line

Project description:We have develped a novel method of making siRNAs (named pro-siRNA for prokaryotic siRNA). To evaluate off-targeting of pro-siRNA, we compared the mRNA expression profiles of HeLa-d1EGFP cells transfected with 4 nM EGFP siRNAs and pro-siRNAs by microarray. We used microarray to study the off-target effect of siRNAs in the HeLa-d1EGFP cell line. After transfection of siRNAs for 24 hrs, RNA were extracted using Trizol. Deep sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #E7530).

2013-02-12 | E-GEOD-44256 | biostudies-arrayexpress

RNAi profiling by array of human CD34-derived erythroid progenitors treated with BCL11A siRNAs

Project description:Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the beta-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here we examine BCL11A as a potential regulator of HbF expression. The high HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the beta-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in beta-hemoglobin disorders. BCL11A siRNA label: B, NT siRNA label: N Experiment Overall Design: Microarray expression analysis from CD34-derived erythroid progenitors treated with either non-targeting (NT) control siRNAs or BCL11A targeting siRNAs. Six samples from the NT control and six samples from the BCL11A siRNA treatment are included. Cells were harvested on day 7 of erythroid differentiation after introduction of siRNAs on day 0 of the differentiation protocol. Experiment Overall Design: 6 BCL11A siRNA datasets, 6 control (NT) datasets

2009-01-13 | E-GEOD-13284 | biostudies-arrayexpress

Gene expression data from cultured mouse cortical (mCTX) neurons in different stimulation and knockdown conditions

Project description:Although the induction of C-FOS in the brain has been extensively studied for several decades to date there has been no attempt to identify the targets of C-FOS at a genome wide level, and it was not known how many genes C-FOS activates in a given cell. To identify potential C-FOS target genes, we performed microarray analysis on RNA obtained from mouse cortical (mCTX) neurons infected with lentivirus containing either a control shRNA (targeting firefly luciferase) or c-Fos shRNA that were subsequently depolarized with 0, 1, 3, or 6 hours of KCl. Embryonic day 16.5 cortices from C57BL/6 mice were dissected, dissociated, plated, and subsequently grown in a cell culture incubator that maintained a temperature of 37 degrees C and a CO2 concentration of 5%. On DIV5, lentiviral supernatant containing either a lentivirus expressing a control shRNA targeting firefly luciferase or a lentivirus expressing shRNA targeting c-Fos was applied to cultures for 6hrs with 6ug/mL polybrene. At DIV7, mCTX neurons were silenced and stimulated with KCl for either 0, 1, 3, or 6hrs, and total RNA was purified using Trizol and the RNeasy mini kit from Qiagen. 10ug of labeled cDNA was subsequently hybridized to Affymetrix mouse genome 430 2.0 arrays with affymetrix processing.

2014-09-07 | E-GEOD-60049 | biostudies-arrayexpress

Combining transcriptome and proteome analysis to identify and characterize the RpoS regulon in Pseudomonas fluorescens

Project description:Background: Microorganisms are the major cause of food spoilage during storage, processing and distribution. Pseudomonas fluorescens is a typical spoilage bacterium that contributes to a large extent to the spoilage process of proteinaceous food. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Our previous work revealed that RpoS contributed to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. Results: RNA-seq transcriptomics analysis combined with quantitative proteomics analysis basing on multiplexed isobaric tandem mass tag (TMT) labeling was performed for the P. fluorescens wild-type strain UK4 and its derivative carrying a rpoS mutation. A total of 375 differentially expressed genes (DEGs) and 212 differentially expressed proteins (DEPs) were identified in these two backgrounds. The DGEs were further verified by qRT-PCR tests, and the genes directly regulated by RpoS were confirmed by 5’-RACE-PCR sequencing. The combining transcriptome and proteome analysis revealed a role of this regulator in several cellular processes, including polysaccharide metabolism, intracellular secretion and extracellular structures, cell well biogenesis, stress responses, ammonia and biogenic amine production, which may contribute to biofilm formation, stress resistance and spoilage activities of P. fluorescens. Moreover, in this work we indeed observed that RpoS contributed to the production of the macrocolony biofilm’s matrix.

2019-11-12 | PXD010845 | Pride

Transcription profiling by array of human breast cancer cell line BT-474 with annexin A1 over-expression or silenced with siRNA to demonstrate design of multi-condition experiments

Project description:Multiple-condition experiment was desinged to be any number of conditions in an experiment without replicate observations for microarray and used to identify genes differentially expressed between different pairs of conditions (treatments).<br> In this study we used breast cancer stable cell lines for overexpressing and silencing annexin A1 (ANXA1), which belongs to a family of -dependent phospholipid binding proteins and are preferentially located on the cytosolic face of the plasma membrane. Cell lines overexpressing ANXA1 (MDA_MB-453/cDNA) were generated by introducing retroviral vectors containing ANXA1 cDNA (pBabe/ANXA1 cDNA) into breast cancer cell line MDA-MB-453 (a low expressor of ANXA1). Breast cancer cell line BT-474, a high expressor of ANXA1, was infected with ANXA1 siRNA-plasmid viruses to knockdown ANAXAI expressor (BT-474/siRNA) where nucleotides corresponding to siRNA were synthesized and ligated into the pLNCX retroviral vector [35,36]. We also used a pLNCX/U6 empty vector to infect BT-474 and obtained an empty vector expressor. Therefore, 5 breast cancer cell lines (MDA_MB-453, MDA_MB-453/cDNA, BT-474, BT-474/siRNA, and BT-474/U6) are attributed to two genotypes: MDA_MB-453 and BT-474. MCE was performed for microarray analysis with these 5 breast cancer cell lines, that is, only one sample was drawn from each breast cancer cell line.

2010-06-26 | E-MEXP-2775 | biostudies-arrayexpress

Expression data of human lung adenocarcinoma cell line H441 treated with TTF-1/NKX2-1 siRNA and TGF-beta

Project description:We determined and analyzed the effect of TTF-1/NKX2-1 on Smad3/Smad4 binding sites by ChIP-sequencing. We used expression microarrays to evaluate the effect of TTF-1/NKX2-1 siRNA on TGF-beta-induced gene expressions. H441 cells were transfected with siRNAs and treated with TGF-beta for RNA extraction and hybridization on Affymetrix microarrays. This submission represents transcriptome component of study.

2014-07-27 | E-GEOD-49675 | biostudies-arrayexpress

Expression data from control and COUP-TFII siRNA treated HUVEC cells

Project description:COUP-TFII plays a critical role in angiogenesis during development. It has also been shown to suppress Notch signaling pathway to confer vein identity. However, the downstream targets and the mechanism mediate COUP-TFII function to regulate these processes remain elusive. To identify the downstream targets and the mechanism by which COUP-TFII regulates agiogenesis and vein specification, we knocked down COUP-TFII in HUVEC cells using COUP-TFII specific siRNA and used microarray analysis to identify downstream targets. Interestingly, we found the expression of many genes in the cell cycle pathway and Notch signaling pathway are significantly altered in the COUP-TFII depleted cells. HUVEC Cells were transfected with siRNA (Control or COUP-TFII siRNA) duplexes (50 nM) and total RNA was isolated 48 hours later.

2011-10-27 | E-GEOD-33301 | biostudies-arrayexpress

Preclinical targeted therapy for retinoblastoma using EpCAM Aptamer siRNA chimeras

Project description:siRNAs have played a major role in cancer drug discovery, but their potential is hampered due to off-target effects. Thus, delivery systems like RNA aptamers have been used to enhance the specific delivery of these siRNAs to cancer stem cells. We report the efficacy of three different EpCAM aptamer siRNA chimeras, which were investigated both in vitro and in vivo for their ability to reduce cancer cell progression. Using these chimeras, we demonstrated specific gene knockdown in EpCAM positive cells which ultimately led to the apoptosis. To study the efficacy of these aptamer chimeras in vivo, retinoblastoma xenografts bearing NCC Rb C 51 cells were created for the first time. Systemic administration of these aptamer chimeras reduced tumour growth to about 50%. We further investigated the central Role of PLK1 in Cancer Progression and demonstrated the anti-cancer effects of targeted EpCAM siPLK1 approach. Using SILAC-Mass spectrometry analysis, we showed that silencing PLK 1 gene can lead to p53 mediated cell cycle arrest. Thus, we establish EpCAM-siRNA chimeras as potential markers for targeted anti-cancer applications, which paves a platform for efficient second line of therapies in addition to existing chemotherapy options.

2022-03-02 | PXD008221 | Pride

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